The interaction between vitamin D and extracellular calcium on osteogenic differentiation

While the role of vitamin D in the prevention of rickets in children and osteomalacia in adults has been well demonstrated, its benefit in the treatment of osteoporosis is subject to controversy. Clinical trials of vitamin D supplementation to prevent fractures have been conducted with mixed results and some meta-analyses have indicated limited benefit in reducing fracture risk. The most consistent beneficial effects of vitamin D have been obtained when combined with calcium supplements. 1,25-dihydroxyvitamin D acts on the three major types of bone cells (osteoblasts, osteoclasts and osteocytes) to initiate either catabolic or anabolic actions on bone. To elucidating the potential benefits of vitamin D to bone health, this study examined direct actions of vitamin D metabolites on bone cells focussing on stimulation of in vitro osteogenic differentiation. Two cell culture models, representing immature and mature stage of osteoblasts, were employed to investigate the role of vitamin D on osteogenic differentiation. The regulation of a variety of gene expressions and modulation of mineral deposition by these cells, were used as key readouts. In chapter 3, vitamin D was observed to play an inhibitory role on mineral deposition by the immature calvarial bone-derived osteoblast-like cells (Calvarial cells) but did not exert any suppressive effect on the mature osteoblast/early osteocyte cell line, MLO-A5. Thus the actions of vitamin D appear to be dependent on either the stage of cell maturation or their skeletal origin. The studies using Calvarial cells were expanded in chapter 4 by utilising cells derived from genetically modified mouse lines, including the global vitamin D receptor (VDR) knockout (VDRKO) and the over-expression of VDR in osteocalcin-expressing cells (OSVDR), in comparison to cells derived from wild-type animals. The active hormone form, 1α,25-dihydroxyvitamin D₃ (1,25D), promoted a mature cell phenotype at physiological levels (around 30 pM) dependent on the level of Vdr mRNA. However, in OSVDR cells with high levels of VDR, a pharmacological concentration of 1,25D (1 nM) appeared to stimulate de-differentiation of the osteoblast phenotype by down-regulating the expression of mature osteoblast/osteocyte genes. Enpp1 and Tnap were identified as key genes to modulate mineral deposition in these models. In chapter 5, the cell line MLO-A5 was again utilised, here for studying the interaction between vitamin D and extracellular calcium on osteoblasts. Both endogenous and exogenous sources of 1,25D, either alone or interacting with extracellular calcium, increased mineral deposition and the expressions of maturation-related genes. Extracellular calcium altered vitamin D metabolism by MLO-A5 cells. Again, key genes associated with mineral deposition were Enpp1 and Tnap. Data from this study confirm the stimulatory actions of vitamin D on osteogenic differentiation and identified an interaction with extracellular calcium levels. Mineral deposition was found to be dependent on 1,25D modulation of Enpp1 and Tnap expressions. A highly novel finding was that the extracellular calcium concentration modulates the metabolism of vitamin D and the maturation of these cells. These data help to address the controversy on the actions of vitamin D on osteoblast differentiation and mineralisation and improve our understanding of their biology.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015